Immunogen and Antivenom Against Violin Spider Venom

ABSTRACT

The invention relates to the isolation, characterisation and expression of DNA fragments encoding sphingomyelinases D from three species of  Loxosceles  genus spiders, namely  L. boneti, L reclusa  and  L. laeta,  and the toxoids thereof. The invention also relates to the production of active sphingomyelinases D and the toxoids thereof using recombinant means and to the use of same as an immunogen for the production in vertebrates of antibodies that neutralise the corresponding venom and the respective fragments F(ab′)2. The invention further relates to the use of recombinant sphingomyelinases D as part of an antigen matrix which can be used in the immunopurification of antibodies and the fragments thereof or as part of any diagnostic device used to obtain clinical confirmation that the causal agent of poisoning in a patient is a spider of the  Loxosceles  genus. In addition, the invention includes molecular vectors for the expression of the DNA fragments, strains comprising same, which can express  Loxosceles  sphingomyelinases D, and methods for the expression thereof. 
     
       
         
               
             
                   
               
                 AA 
               
                 Porcentaje de identidad de aminoacidos entre necrotoxinas 
               
                 de diferentes especies de  Loxosceles . 
               
               
               
               
               
               
               
               
               
               
               
             
                 especie 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
                 BB 
                 Lb1 
                 Lr1 
                 Lr2 
                 La 
                 Li 
                 Ll-H17 
                 Ll-H13 
                 Ll-1 
                 Ll-2 
               
                   
               
                 Lb1 
                 — 
                   
                   
                   
                   
                   
                   
                   
                   
               
                 Lr1 
                 91 
                 — 
               
                 Lr2 
                 84 
                 90 
                 — 
               
                 La 
                 86 
                 88 
                 87 
                 — 
               
                 Li 
                 82 
                 84 
                 88 
                 80 
                 — 
               
                 Ll-H17 
                 57 
                 60 
                 60 
                 59 
                 59 
                 — 
               
                 Ll-H13 
                 57 
                 60 
                 61 
                 58 
                 60 
                 80 
                 — 
               
                 Ll-1 
                 56 
                 59 
                 59 
                 58 
                 59 
                 99 
                 80 
                 — 
               
                 Ll-2 
                 57 
                 60 
                 61 
                 59 
                 59 
                 82 
                 94 
                 82 
                 — 
               
                   
               
                 AA . . . AMINO ACID PERCENTAGE OF IDENTITY BETWEEN NECROTOXINS OF DIFFERENT  LOXOSCELES  SPECIES 
               
                 BB . . . SPECIES

TECHNICAL FIELD

This invention refers to the recombinant proteins that comprise thesequence of sphingomyelinase D, the main component of the venom of theviolin spider (Loxosceles boneti; Loxosceles reclusa, and Loxosceleslaeta [Peruvian variety]). When they are injected into mammals, theproteins generate an effective immune response to neutralize the toxicaction of the whole venom of those arachnids. It also refers to the useof these proteins as immunogens for the production in vertebrates ofantibodies against the whole venoms of said arachnids. It also refers tothe composition of said antibodies or the antigen-binding fragmentsthereof and their use for the treatment of violin spider bite poisoning,and to an antigenic matrix capable of specifically binding neutralizingantibodies of the arachnid's venom, useful for the purification byimmunoaffinity of such sero-therapic and fab-therapic agents.Additionally, it refers to a diagnostic device that incorporates saidmatrix to determine the species of the spider causing the poisoning, andto the method for performing said diagnosis. In another scope of theinvention, the DNA fragments that codify the respective recombinantproteins and the expression constructions for said fragments, as well asthe bacterial cells transformed with said constructions and the methodfor producing the proteins using recombinant means are also included.

BACKGROUND

Spiders pertaining to the Loxosceles genus are commonly known as violinspiders because they have a violin-shaped mark with the neck pointingbackwards in the anterosuperior part of the cephalothorax (Platnick,2000).

Spiders of this genus are found all over the world, generally intropical and temperate climate regions (Ramos, 2000). In Mexico, thereare about 39 species of this genus (Hoffman, 1976; Gerstch, 1983). Intheir natural habitat, they can be found under the bark of trees, underrocks and in caves. They can be found in coexistence with human beings:under furniture, in the corners of rooms, in cracks, grooves oflivestock facilities, wood, bricks, and abandoned waste. One of the maincauses for Loxosceles bite accidents is, precisely, the constantcoexistence with man.

Poisoning caused by the bite of spiders of the Loxosceles genus iscalled LOXOSCELISM. The violin spider bite commonly produces localnecrotic lesions or dermonecrosis (necrotic Loxoscelism), while in somecases it can cause non-necrotizing systemic effects (systemicLoxoscelism).

The extent of local necrosis is related to the spider's stage in ofdevelopment, the dose of venom injected in the bite and the immune stateof the patient (Moye de Alba, 1997; Maguire, 1998).

Dermonecrosis is preceded by edema, the accumulation of inflammatorycells and vasodilatation, all of which culminates in a black vesiclecommonly called “bull's eye” lesion. Sometimes the Loxosceles genus mayalso produce intravascular hemolysis associated with spherocytosis, acondition that persists for several days (Maguire, 1988; Rosse, 1998).

In Mexico, 15 cases of violin spider bite poisoning were treated in theSocial Security Institute (personal information, Dr. María del CarmenSánchez, “La Raza” Hospital, Mexico City) during the past 5 years, 11 inadults and 4 in children. In 53.3% of the cases, in addition to thenecrotic loxoscelism, there was systemic loxoscelism, and 62% of thecases where both occurred, the patients died.

Biochemistry of the Venom

Until now, few spider venoms have been studied in detail. The Loxoscelesvenom is composed of at least ten to twelve components (Russell, 1987),among them: sterases, alkaline phosphatase, hyalouronidase,phosphohydrolases, lypases and proteases, among others. It has so farbeen proven that the main component, and the one that causesdermonecrosis, is sphingomyelinase D (SMD). This enzyme binds to thecell membranes (epithelial, endothelial of the vascular tissue and redblood cells) hydrolyzing sphingolipids to subsequently releasephosphoceramide and choline (Gatt, 1978). The hydrolysis induces thechemotaxis of neutrophils causing vascular thrombosis and an Arthus-typereaction (Moye de Alba, 1997; Maguire, 1998; Sánchez, 1993).

Hyaluronidases, other enzymes involved in poisoning, are common in thepoisons of almost all spiders (Tan and Ponnundarai, 1992). They havebeen detected in a considerable number of species (Geren, 1984)including Loxosceles sp., although in these last species, very lowenzymatic activity has been reported (Wright, 1973). Hyaluronidases areconsidered venom-dispersion factors, because the hydrolysis of thehyaluronic acid facilitates the diffusion of the other toxic componentswithin the victim's tissues (Cevallos et al., 1992). Hyaluronidases actas a dispersing agent and it is thought that proteases might be directlyinvolved in dermonecrosis through the digestion of the proteins thatform the extracellular matrix (Young, 2001).

Recent studies have identified two proteases, Loxolysine A andLoxolysine B, in L. intermedia. Loxolysine A is a 20-28 kDametalloprotease with fibrogenolytic activity (degrading fibrinogen) andfibronectinolytic activity (degrading fibronectin). This protein mightbe involved in the local hemorrhagic effects observed at the site of thebite and in some cases in the systemic hemorrhages, while Loxolysine Bis a 32-35 kDa protease with gelatinolytic activity, and although itsfunction is as yet unknown, it may possibly participate in thedegradation of collagen within the extracellular matrix (Feitosa et al.,1998).

Three isoforms (P1, P2, and P3) of the necrotoxic fraction have beenfound in L. intermedia; they were extremely similar to each other at thebiochemical and immunological level. The first two are necrotoxic, P2with a greater effect, while P3 was completely inactive. The analysis ofthe amino acidic sequences of the first 35 amino acids of the extremeamino terminus of the isoforms revealed that they were identical in manyways. They were also compared with the partial sequences of the toxinsof other previously reported Loxosceles species, obtaining a high degreeof similarity (Tambourgi, 1998).

In 1968 Smith and Micks demonstrated that the injection of L. reclusa,L. laeta or L. rufuscens venom into rabbits produced similar necroticreactions. Recent studies compared the amino terminus sequences ofsphingomyelinase of L. reclusa, L. deserta, L. gaucho, L. intermedia andL. laeta venom determining that they were homologous among them (Barbaraet al., 1996B).

To date, the complete sequences of the sphingomyelinase D of only 2 ofthe Loxosceles species have been reported—L. laeta (Fernandes Pedrosa etal., 2002) and L. intermedia (Kalapothakis et al., 2002); they show anidentity of barely 59% between them. Only the first 34 AAs of thesphingomyelinase of L. reclusa are known and they happen to have an85.7% similarity with the equivalent sequence of L. intermedia and 60%with that of L. laeta; for this reason it is impossible to establish aprobable sequence for the almost 244 AAs of the enzyme that are stillunknown. Similarly, only 35 AAs of the amino terminus region of thesphingomyelinase D of L. deserta and 39 of that of L. gaucho are known(for L. deserta only the sequence derived from the gene is known).

Work on the generation of antibodies against particular species ofLoxosceles and cross tests of the same with venoms of spiders of otherspecies of this genus have been reported. For example, a set ofmonoclonal antibodies against the dermonecrotic component (of 35 Kda) ofthe venom of L. gaucho was developed, which, while being effective inrecognizing and neutralizing the homologous venom, was far less able torecognize the venoms of L. laeta and L. intermedia. Its neutralizingcapacity was almostnil, compared with the polyclonal antibodiesgenerated against the same component of L. gaucho which adequatelyrecognized and neutralized the venom of L. intermedia, and partially(60%) recognized that of L. laeta, suggesting the presence of differentepitopes in the dermonecrotic components of these species, as well asdifferences in the composition and toxicity of these venoms (Guilhermeet al., 2001). Moreover there is evidence of a marked cross-reactivitybetween the venoms of L. reclusa and L. deserta when the venom of eitherof the two species is used to generate antibodies (Gomez et al., 2001).

In general terms, there are two approaches for the treatment/preventionof poisoning by poisonous animals such as the violin spider: passiveimmunization (by means of sero-therapic and fab-therapic agents) andactive immunization (through vaccines); the former is a therapeuticmeasure, while the latter is rather a preventative measure.

Both venoms and isolated toxins have been used to generate vaccines.However, exposure to most venoms does not result in protective immunity.Furthermore, all attempts to create protective immunity against venoms,such as vaccines, have failed (Russell, 1971). In contrast, success hasbeen obtained creating this type of immunity against individual toxins,including vaccines against diphtheria (Audibert et al., 1982), tetanus(Alouf, 1985), the toxoid of α-Latrotoxin (Alagón et al., 1998) andsphingomyelinase D of L. laeta (Araujo et al., 2003).

Passive Immunization

Aside from the palliative treatment of some of the specific symptom, theonly treatment available for poisoning is passive immunization.

In the case of passive immunization, the antibodies or their fragmentsthat will bind to the venom (antigen) are exogenous; i. e., they areproduced in a first animal. The serum or antivenom from the first animalis then administered to the individual already affected by poisoning(host) to provide him with an immediate and active source of specificand reactive antibodies. The administered antibodies or their fragmentswill work then, in some sense, as if they were endogenous antibodies,binding the toxins of the venom and neutralizing their toxicity.

Depending on their final use, commercial generation of antivenoms can beundertaken, in various mammals such as mice, rabbits, goats, cows andhorses, the horse being the animal of choice of most laboratories sinceit is sturdy and tolerates the immunization process and especiallybecause it produces high outputs (up to 16 L per bleeding).

However, there are some technical disadvantages to using horses for theproduction of antivenoms, among them, the need for large amounts ofvenom (immunogen or antigen) for performing immunization, forcing thelaboratories to have large arachnariums or to contract out the work ofgathering large collections of specimens in order to have sufficientquantities of venom available. For example, it is estimated that theproduction, evaluation and quality control of a lot of antivenom inhorses requires highly standardized venom from five thousand spiders,which limits its commercial feasibility. Therefore, having recombinantimmunogens capable of triggering an immune response comparable to thattriggered by the administered venoms may be a significant alternativefor the production of antivenoms, since stable and consistent immunogenswould be produced in sufficient quantities at significantly lower costsand with fewer risks than those incurred by keeping arachanariums or theimpact of massive collections on ecosystems.

In particular, there are two reports about the use of recombinantproteins as immunogens for the generation of antibodies against spidervenoms in mammals, namely, the toxoid of α-Latrotoxin (Alagón et al.,1998) and a fusion protein that comprises the sequence ofsphingomyelinase D of Loxosceles intermedia (Araujo et al., 2003).

In Mexico and Latin America, one of the main producers of antivenomsagainst the venoms of snakes and spiders (scorpions and black widowspiders) is the Instituto Bioclón, S.A. de C.V., which producesantibodies in horses and later purifies and hydrolyzes them in such away that their antivenoms are in fact F(ab′)₂ fragments of theantibodies, i.e., they are fabotherapics. Specifically, they produceantivenom against the venom of the black widow spider, Aracmyn®.

Due to the variety of common and serious side effects of non-purifiedantivenoms, the physician must be extremely careful to avoid givingexcessive amounts of equine products. A generally accepted theory isthat the high incidence of side effects from the current horseantivenoms is caused by the excess of irrelevant protein they contain(irrelevant in the sense of not having a specific activity against thevenom). According to this theory, the removal of that irrelevant proteincould reduce the exogenous protein charge applied to the body and as aconsequence, reduce the incidence of adverse immune responses.

Some researchers in the state-of-the-technique have considered thepossibility of purification by immunoaffinity. Most of those studieshave only tested antibodies against a single toxin; for example, Yang(1977) proved the purification by immunoaffinity of antibodies against asnake venom toxin. This researcher used cobratoxin, a neurotoxic proteinisolated from the venom of the Taiwan cobra (Naja naja atra), bound toSepharose, as an antigenic matrix and used formic acid to elute thetoxin-specific antibodies. The antibodies thus purified were reported tohave greater ability to neutralize the toxin than the non-purifiedserum.

Other researchers have been following similar purification schemes, suchas Kukongviriyapan et al. (1982), who used the toxin 3 of Naja najasiamensis bound to several materials to form antigenic matrixes,obtaining a separation of horse-specific antibodies; Ayeb and Delori(1984), who also followed Yang's scheme to purify antibodies againstscorpion-specific toxins; and Lomonte et al. (1985), who purifiedantibodies against the myotoxin of B. asper coupled to Sepharose.

Thus again, having recombinant sphingomyelinases that can specificallybind, preferably bound to an inert matrix, only those antibodies ortheir fragments that have a high specificity towards the necrotoxiccomponent of the violin spider venom, may be of great help for removingthe irrelevant protein for the treatment of poisoning, significantlyreducing the risk of adverse immune reactions.

After an incident with Loxosceles, it is not uncommon for the treatingphysician, or even the affected patients or his/her parents in the caseof children to, wrongly assume that it is the bite of an insect or ofanother type of spider, therefore applying insufficient or wrongtreatment to the patient. By the time the unequivocal symptoms ofloxoscelism start to appear, the necrosis of the tissue surrounding thewound may be quite advanced and can only be corrected using a skingraft. In this sense, it would be convenient to have an easily readablediagnostic system that allows the treating physician to determine duringthe first hours after the incident whetherit is indeed a violin spiderbite and to immediately start an effective treatment that prevents thedevelopment of necrosis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chromatogram of gel filtration of molecular exclusion (SephadexG-75) of gland extract of L. boneti. The roman numerals correspond tothe different fractions obtained. The red line corresponds to absorbanceat 260 nm and the blue one at 280 nm.

FIG. 2. Analysis through SDS-PAGE of the fractions obtained by gelchromatography. 1) Molecular weight markers. 2) Fraction I (2.24 μg). 3)Fraction II (3.84 μg). 4) Fraction III (1.44 μg). 5) Fraction IV (8 μg).6) Lysate of L. boneti glands (28 μg).

FIG. 3. Results of Cationic Exchange Chromatography for fraction II ofL. boneti. Each peak represents an isoform. The arrows show otherpossible isoforms found in lesser amounts within fraction II. The columnwas of the Mono S type. The column was 5 cm long by 0.5 cm in diameter.The flow rate was 1 mL/min. Sensitivity was of 0.2 AU. The buffer usedwas 20 mM ammonium acetate pH 4.7, using a gradient of 0-2 M NaCl. Theplotting rate was of 15 cm/hr.

FIG. 4. Alignment of sequences of the 5 proteins with SMD activities,which comprised the recombinant proteins of this invention, with othersreported in the literature is shown.

Access number in the Gene Bank: species *Lb1. AY559844 L. boneti *Lr1.AY559846 L. reclusa *Lr2. AY559847 L. reclusa *Ll1. — L. laeta *Ll2. —L. laeta La. AAP44735 L. arizonica Li. AAQ16123 L. laeta Ll.H17.AAM21154 L. laeta LlH13. AAM21155 L. laeta *This patent

FIG. 5. The percentages of amino acid identity among thesphingomyelinases D of several species of Loxosceles are shown.

Access number in the Gene Bank: species *Lb1. AY559844 L. boneti *Lr1.AY559846 L. reclusa *Lr2. AY559847 L. reclusa *Ll1. — L. laeta *Ll2. —L. laeta La. AAP44735 L. arizonica Li. AAQ16123 L. laeta Ll.H17.AAM21154 L. laeta LlH13. AAM21155 L. laeta *This patent

DETAILED DESCRIPTION Definitions.

The term “antibody” is used to refer to polyclonal antibodies and theirfragments.

The term “fragment”, when referring to antibodies, comprises a portionof the whole antibody, generally the fragment of antigen binding, e.g.fragments Fab, Fab′, F(ab′)₂, and Fv.

The terms “neutralize” or “neutralizing” or “neutralizing antibodies”refer to the ability of the antibodies of this invention to bind to thesphingomyelinase D of spiders of the Loxosceles genus, whether isolatedor as part of the whole venom of those spiders, and to cancel its toxiceffect and that of the said venom.

The term “treatment” refers to therapeutic treatment. Those that needtreatment include those individuals bitten by one or more spiders of theLoxosceles genus.

The term “toxoid” refers to a mutant version of the recombinant proteins(recombinant SMD) object of this invention that lack enzymatic anddermonecrotic activity, but that retain the property of generatingantibodies that neutralize the venom of the Loxosceles spider when usedto immunize vertebrates, and in particular, mammals.

The term “pharmaceutically acceptable carrier” refers to a solid orliquid excipient, diluent or substance that can be used safely forsystemic or topic administration. Depending on the particular route ofadministration, a variety of pharmaceutically acceptable carrierswell-known in the state of the art includes solid or liquid excipients,diluents, hydrotopes, surface active agents, and encapsulatingsubstances. The amount of carrier used in conjunction with theantibodies or their F(ab′)₂ fragments provides a practical manageableamount of material by unitary doses of the composition.

Acceptable carriers for systemic administration that can be incorporatedinto the composition of this invention include sugar, starches,cellulose, vegetable oils, buffers, polyoles, and alginic acid. Thespecific pharmaceutically acceptable carriers are described in thefollowing documents, which are hereto incorporated by reference: U.S.Pat. No. 4,401,663 Buckwalter et al., issued Aug. 30, 1983; Europeanpatent application No. 089710, LaHann et al., published Sep. 28, 1983;and European patent application No. 0068592, Buckwalter et al.,published Jan. 5, 1983. The preferred carriers for parenteraladministration include propylene glycol, pyrrolidol, ethyl oleate,aqueous ethanol and combinations thereof.

Representative carriers include acacia, agar, alginates,hydroxyalkylcellulose, hydroxypropyl-methyl-cellulose,carboxymethylcellulose, carrageenin, powdered cellulose, guar gum,cholesterol, gelatin, agar gum, gum Arabic, gum karaya, ghatti gum,carob gum, octoxinot 9, allylic alcohol, pectin, polyacrylic acid andits homologues, polyethylenglycol, polyvinylic alcohol, polyacrylamide,sodium lauryl sulphate, polyethylene oxide, polyvinylpirrolidone, glycolmonostearate, propylenglycol monostearate, xanthan gum, tragacantum,sorbitan esters, estearylic alcohol, starch and its modifications. Theappropriate ranges vary from around 0.5% to around 1%.

As can be seen from the aforementioned, unlike the problem of poisoningby the black widow spider (Latrodectus mactans), for which there arealready some treatments using antivenoms, (F(ab′)₂ fragments of horsepolyclonal antibodies), for loxoscelism, there is still no commercialtreatment. The generation of antivenoms by immunization with the venomof the spider could be an alternative; however, it has the disadvantageof requiring a large number of spiders (about 5,000 per batch) toextract enough amounts of the venom. Also, its use implies that theserum produced by the animal contains large amounts of antibodiesagainst the varied proteins of the venom, most of them have eitherminimal or no effect on the poisoning process in mammals, and thereforethe antibodies against those proteins have little value for treating thepoisoning and instead, they contribute large amounts of proteins thatare exogenous to the organism being treated, potentially causing severeside effects. In general, some approaches that have been followed toreduce that amount of protein have been separating the immunoglobulinfraction of the serum, eliminating other serum proteins (such asalbumin) by precipitation with ammonium or sodium sulphate, with thecorresponding decrease of the neutralizing activity, and hydrolyzingthat fraction with tripsin or pepsin to release the F(ab) fragments orF(ab)₂, that preserve the neutralizing activity. However, the antibodyfraction or their fragments continue to have a high proportion ofirrelevant protein (because they are antibody fragments against all thecomponents of the whole venom and against multiple undefined antigens).

This problem could be resolved through immunoaffinity purification ifthere were an immunogen matrix that allowed for the specific separationof the source from the immunoglobulins or from the fragments produced bytheir hydrolysis, those specific antibodies or fragments, responsiblefor neutralizing the toxic effect of the whole venom.

On the other hand, as mentioned before, there is a need for a reliablediagnostic system that allows the attending physician to determinequickly and easily whether he is dealing with a Loxosceles bite, andthen to be able to immediately administer an appropriate treatment thatprevents the necrosis of the tissue surrounding the area of the bite.

In order to solve these problems, the inventors of this invention haveundertaken to characterize the venom of L. boneti, isolating andpurifying its dermonecrotic component and to isolate and characterizeits codifying DNA, as well as the codifying DNA of the dermonecroticcomponent of L. reclusa and L. laeta (Peruvian variety), to clone thecodifying sequences into appropriate expression vectors, to express theactive recombinant proteins, and test them regarding their ability togenerate an effective immune response for the neutralization of thenecrotoxin and the whole homologue venom, as well as in cross-reactiontests of the venom of these species of Loxosceles.

The active recombinant proteins of L. boneti (SMDrLb), L. reclusa(SMDrLr), and L. laeta (Peruvian variety) (SMDrLI) may be used asimmunogens to generate antivenoms in vertebrates; bound to solid orsemisolid substrates to generate antigenic matrixes useful forimmunopurification of antivenoms; and in the design and construction ofdiagnostic systems to determine if a patient suffers from loxoscelism.

As shown in Example 1, the inventors of this invention separated thewhole venom of L. boneti into fractions, finding that only fraction IIshows dermonecrotic activity. They were able to separate 3 isoforms ofthe dermonecrotic component. They found that only isoforms I and II areactive, the former being the more active one, and they obtained an aminoacidic sequence of the amino terminus of the three isoforms, which wereused to design the specific oligos for obtaining the clones of thenecrotoxic components from the RNAm extracted from the venom glands ofthe spiders (see Example 3), clone 30-8 being the one selected for itsexpression. For this purpose, clone 30-8 was subcloned in thepTrcHIS-TOPO plasmid, obtaining pTrcHIS-TOPO 30-8 plasmid (see detailsin Example 4) and expressed in E. coli XL1 Blue, obtaining itsexpression in the form of inclusion bodies that were solubilized andrefolded. The solubilized and refolded recombinant protein lackedsphingomyelinase D (SMD) and dermonecrotic activity and the antibodiesgenerated in rabbits against it; although capable of recognizing theprotein through Western Blot, they were not able to neutralize the wholevenom of the L. boneti spider.

After an analysis, the following factors were taken into considerationin an effort to express the recombinant protein in a soluble and activeform: (i) the sequence of the fragment used lacked the first 4 codons,hence the amino acidic sequence of the recombinant protein lacked thefirst 4 amino acids of the extreme amino terminus; (ii) just before theBamH I cut site, vector pTrcHIS-TOPO has the 36 AA sequence, includingthe initial methyonine and a tail of 6 histidines useful for therecovery of the protein which, plus the 2 codons occupied by the BamH Isite, add a total of 38 amino acids to the recombinant protein, whichamino acids could be responsible for its poor plication, and (iii) it isknown (see pET System Manual) that certain strains of E. coli may favorthe adequate plication of some the recombinant proteins they express.Additionally, the successful expression of the recombinant proteincomprising the amino acidic sequence of one of the isoforms ofsphingomyelinase D of L. laeta in E. coli BL21 had been reported(Fernandes Pedroza et al., 2002).

Therefore it was decided to complete the codifying sequence (the 4missing amino acids) as shown in Example 5; changing to the pQE-30expression vector that only adds 12 amino acids to the recombinantprotein at its extreme amino terminus, and expressing it in the BL21strain of E. coli (see Example 6). Again, an expression in the form ofinclusion bodies was obtained which, although considerable, hadnegligible enzymatic activity.

It is known that if the expression is too high, all the proteinaccumulates in the form of inclusion bodies, thus it may be advisable totry to reduce the protein expression. Whit this finality, a controlledexpression of the protein was performed, as detailed in Example 7, thistime managing to express the protein both as inclusion bodies andsoluble. Once the protein was purified was proved to have the samespecific activity as have the native isoform I, and with an LD50 of 2.55μg per mouse.

The soluble recombinant sphingomyelinase D of L. boneti (SMDrLb) thusproduced was used to inoculate rabbits (see Example 8) obtaining titlesof up to 29,300. The resulting antibodies were neutralizing for both theactive recombinant protein (ED50=154.6 μL/mouse for 7.8 μg of theantigen), and the native venom of L. boneti (ED50=149.6 μL/mouse with 3LD50 of the native venom).

Based on the experiment followed to obtain the sphingomyelinase D (thedermonecrotic component) of L. boneti, we proceeded to obtain thesphingomyelinase D of L. reclusa and L. laeta (Peruvian variety). In thefirst case, the amino acidic sequence of the first 34 amino acids of theamino terminus of the sphingomyelinase D of L. reclusa is known from theliterature (Barbaro, K. C. et al., 1996), and upon exact coincidence ofthe first 5 amino acids with those corresponding to L. boneti, wedecided to use the same oligos to obtain the cDNA of the dermonecroticcomponent from the RNAm extracted from the glands of the spider (seeExample 9). In this case, the cDNAs of two active isoforms of the SMD ofL. reclusa were obtained, SMDrLr1 and SMDrLr2 (with 90% of similaritybetween them) with an activity of 27.2 U/mg for SMDrLr1 and 11.47 U/mgfor SMDrLr2. As was the case with L. boneti, the inventors subcloned theclones in the pQE-30 plasmid, and they were expressed in a controlledway in the BL21 strain of E. coli (see Example 10). To exemplify theiruse as immunogens for generating neutralizing antibodies from the venomof L. reclusa spiders, one of the soluble and active recombinantsphingomyelinases D of L. reclusa thus produced, SMDrLr1, was used toinoculate rabbits (see Example 11), resulting in titres of up to 33,000.The resulting antibodies were neutralizing for both the activerecombinant protein (ED50=165 μL/mouse for 12 μg of the recombinantprotein), and the native venom of the L. reclusa spider (ED50=175μL/mouse with 12 μg of the venom).

In the second case, the complete amino acidic sequence of some of theactive isoforms of sphingomyelinase D of L. laeta (Brazilian variety) isknown from the literature (Fernandes Pedrosa et al., 2002). Based onthem, the Ll5′Bam HI and Ll3′Sal I oligos were designed to obtain thecDNA of the dermonecrotic component from the RNAm extracted from theglands of the spider (see Example 12). In this case, the inventorssucceeded in isolating the cDNA of 2 distinct and active isoforms: theformer, SMDrLl1, with an activity of 58.43 U/mg, and the latter,SMDrLl2, with an activity of 252 U/mg. As was the case with L. bonetiand L. reclusa, the inventors subcloned the clones in the pQE-30plasmid, and they were expressed in a controlled way in the BL21 strainof E. coli (see Example 13). When the codifying DNA fragments weresequenced, one of the codified proteins, SMDrLl1, showed a primarysequence slightly different (99% of similarity) from that reported byFernandes Pedrosa et al. for isoform H17 (which is the isoform hereports as active), while SMDrLl2, which in this invention proves tohave almost 3 times the activity of SMDrLl1, showed a sequence 94%identical to isoform H13 that is reported by Fernandes Pedrosa et al. asinactive. To exemplify their use as immunogens for generatingneutralizing antibodies from the venom of the L. reclusa spider, one ofthe soluble and active recombinant sphingomyelinase D of L. laeta(Peruvian variety) thus produced, SMDrLl1, was used to inoculate rabbits(see Example 14), obtaining neutralizing antibodies both from the activerecombinant protein (DE50=200 μL per mouse for 12 μg of protein) andfrom the native venom of the L. laeta spider (Peruvian variety)(DE50=225 μL per mouse of venom).

The recombinant proteins of this invention, SMDrLb, SMDrLr1, SMDrLr2,SMDrLl1, and SMDrLl2, were expressed using the pQE30 plasmid, which addsa sequence of 12 amino acids—MRGSHHHHHHGS (SEQ. ID. NO: 25) that includea tail of 6 histidines—to the amino region (amino versions) of theprotein. However, they can be expressed in other expression systems thatintroduce other amino acid sequences to either the amino terminus or thecarboxyl of the proteins. One example is pQE60 plasmid (INVITROGEN),which includes only the Methionine, Glycin and Serine amino acids in theamino region while in the carboxyl terminus it includes an 8-amino acidsequence RSHHHHHH (SEQ. ID. NO: 26). In Example 16, the Lb1, Lr1, Lr2,and Ll2 clones were subclonated in the pQE60 plasmid and were expressedin a controlled way to obtain the carboxyl versions of the proteins(with the Histidine tail in that terminus). The recombinant proteinsobtained, which like the corresponding amino versions comprise thesequences SEQ.ID.NO:11, SEQ.ID.NO:13, SEQ.ID.NO:15, and SEQ.ID.NO:21,were active.

One scope of this invention, then, refers to the SMDrLb, SMDrLr1,SMDrLr2, SMDrLl1, and SMDrLl2 recombinant proteins, either their aminoversions (with the Histidine tail at the amino terminus) or theircarboxyl versions (with the Histidine tail at the carboxyl terminus),that comprise the native sphingomyelinase D sequences of L. boneti, L.reclusa and L. laeta spiders, SEQ. ID. No: 11, SEQ. ID. No: 13, SEQ. ID.No: 15, SEQ. ID. No: 19, and SEQ. ID. No: 21 respectively or variants ormutants of those sequences, and that show sphingomyelinase D activity.

Another scope of this invention refers to the DNA fragments thatcomprise the codifying sequences of the recombinant proteins SMDrLb,SMDrLr1, SMDrLr2, SMDrLl1, and SMDrLl2, with the SEQ. ID. No: 10, SEQ.ID. No: 12, SEQ. ID. No: 14, SEQ. ID. No: 18, and SEQ.ID.No: 20,respectively; its obtaining is detailed in Examples 5, 9, and 12.

It is known that the genetic code is degenerated, i.e., for one aminoacid there is usually more than one codifying codon; generally, thedifference among these codons is at the third base position. It isobvious to an expert in the state of the technique that it is possibleto perform substitutions of some bases in any of the codifyingnucleotidic sequences of the recombinant proteins of this invention(SMDrLb, SMDrLr1, SMDrLr2, SMDrLl1 and SMDrLl2), that codify exactly thesame amino acidic sequences as those present in SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 21, generating“silent mutations” of those sequences. This can be especially usefulwhen one wants to express the recombinant proteins of this invention invarious recombinant hosts, since it is known that different kinds ofhosts have a “preference” of use towards certain codons for particularamino acids. Such “silent mutations” fall within the scope of thisinvention since the products of their expression are once again the samerecombinant proteins SMDrLb, SMDrLr1, SMDrLr2, SMDrLl1 and SMDrLl2 ofthis invention.

Moreover, it is also obvious for an expert in the state of the techniquethat it is possible to substitute some amino acids outside the activesite of the protein for others of the amino acidic sequences withsimilar characteristics; e.g., one polar amino acid for another polaramino acid, an aromatic one for another aromatic one, a charged one foranother, etc. It is not expected that such mutations produce significantchanges in the activity of recombinant proteins, hence those point- orsite-specific mutations for the codifying DNA fragments of therecombinant proteins of this invention that produce functionallyequivalent proteins (i.e., those that have sphingomyelinase D activityand that cause dermonecrosis in a mammal inoculated with them) areincluded within the scope of this invention.

An additional scope of this invention refers to mutants of therecombinant proteins object of this invention and their codifying DNAs,which constitute toxoids of those proteins, i.e., they lack enzymaticactivity as sphingomyelinase D and the dermonecrotic effect, but theyretain the property of being expressed in a soluble form with themethods described above and of generating antibodies, that neutralizethe venom of Loxosceles spiders by inoculating vertebrates—especiallymammals.

Particularly based on the description of the active center of one of theisoforms of sphingomyelinase D of L. laeta (Murakami et al., 2005),where it is made evident that the binding to the substrate and thetransition state of the enzyme are stabilized by the Mg²⁺ ion, whichforms a coordination binding with the Glu32, Asp34, and Asp91 aminoacids and molecules of the solvent.} The amino acids His12 and His47play a key role in the acid-base catalytic mechanism proposed by theauthors (all of the amino acids are counted from the extreme aminoterminus of the mature protein). Therefore, those 5 amino acids prove tobe especially attractive blanks for performing a directed mutagenesismethod to try to obtain mutants that are toxoids of thesphingomyelinases D of Loxosceles spiders. In L. boneti and L. reclusa,the equivalent of these amino acids are Glu31, Asp33 and Asp91, and theHis 11 and His47 histidines. Special attention was given to the ideathat effecting the mutations on Glu32 (or Glu31 for L. boneti or L.reclusa) would be highly attractive for performing the substitution ofan amino acid that breaks the coordination bond with the Mg²⁺ ion, andon His12 (or His11 for L. boneti or L. reclusa) it would be highlyattractive for performing the substitution of an amino acid that,without interfering with the dimerization of sphingomyelinase D, makesit lose its enzymatic activity.

To prove this, by way of illustration but without limitation, as shownin Example 18, a toxoid for the recombinant protein SMDrLb was built,substituting Histidine 11 with Lysine and generating the SEQ.ID.No: 27that codifies for the amino acidic sequence SEQ.ID.No:28.Simultaneously, a toxoid for the same SMDrLb recombinant protein withthe Glu31 substituted by Lysine was built, generating the SEQ.ID.No:29that codifies for the amino acidic sequence SEQ.ID.No:30. Thesemutations cause the proteins (SMDrLb(H11K) and SMDrLb(E31K),respectively) expressed by the above-mentioned methods to lack enzymaticactivity as sphingomyelinase D and the dermonecrotic effect, but toretain the property of being expressed in a soluble form with themethods described above and of generating, by inoculation ofvertebrates—particularly mammals—, antibodies that neutralize the venomof the Loxosceles spider (at least that of L. boneti).

Another scope of this invention refers to the expression vectors inwhich are cloned the DNA fragments that comprise any of the followingsequences: SEQ. ID. NO: 10, SEQ. ID. NO: 12, SEQ. ID. NO: 14, SEQ. ID.NO: 18, SEQ. ID. NO: 20, SEQ. ID. NO: 27, and SEQ. ID. NO: 29. There isa great variety of expression vectors so when selecting them, it isimportant to consider that the number of amino acids added to therecombinant proteins not to be so high as to drastically reduce thesolubility and/or activity of the expression product. Some examples ofexpression vectors are pQ30 and pQ60 by Qiagen. Obviously anotherelement to consider during selection is the bacterial strain to be used.

Another scope of this invention refers to the recombinant bacterialstrain to be used in the expression of the recombinant proteins objectof this invention. Escherichia coli is the best known bacterial speciesand is widely used for the expression of recombinant proteins, hence therecombinant bacterial strain of this invention is preferably from thisspecies. As has been previously mentioned, some strains favor theplication of the recombinant proteins that they express. An example ofthis kind of strain is Escherichia coli BL21. Thus the recombinantbacterial strain of this invention is preferably E. coli BL21.

Another scope of this invention refers to the recombinant methods toproduce the SMDrLb, SMDrLr1, SMDrLr2, SMDrLl1, SMDrLl2 proteins or theirtoxoids through the use of DNA fragments with the sequences SEQ. ID. No:10, SEQ. ID. No: 12, SEQ. ID. No: 14, SEQ. ID. No: 18, SEQ. ID.No: 20,SEQ. ID. NO: SEQ. ID. NO: 27, and SEQ. ID. NO: 29, respectively. This isillustrated in Examples 7, 10, and 13. Thus a general method for theproduction of the recombinant proteins object of this invention iscomprised of the following steps:

(a) incubating in an adequate medium and in adequate culture conditionsa transformed recombinant bacterial strain with an expression vectorthat comprises a DNA fragment selected from the group that consists ofDNA fragments with the SEQ. ID. NO: 10, SEQ. ID. NO: 12, SEQ. ID. NO:14, SEQ. ID. NO: 18, SEQ. ID. NO: 20, SEQ. ID. NO: 27, and SEQ. ID. NO:29 sequences.

(b) optionally, separating the cellular mass from the medium;

(c) breaking the cells to release the protein, and

(d) optionally, separating and purifying the recombinant protein.

The selection of the vector-host expression system may be varied. Inthis invention, by way of illustration but without limitation, the BL21strain of E. coli was selected to be used in the production methods ofthe recombinant proteins of this invention; a controlled expression ofthem was made by means of an IPTG 0.1 mM induction, so that preferably,when an adequate cellular mass is reached using the method for producingthe recombinant proteins object of this invention, the expression isinduced with the concentration of IPTG of 0.1 mM at a temperature ofbetween 20 and 25° C. for at least 12 to 20 hours.

Another scope of this invention refers to the use of the recombinantproteins SMDrLb, SMDrLr1, SMDrLr2, SMDrLl1, SMDrLl2, SMDrLb(H11K) andSMDrLb(E31K) as immunogens to generate neutralizing antibodies of thewhole venom of Loxosceles genus spiders in vertebrates with the purposeof industrially producing antivenoms against the venom of those spiders.This is clearly illustrated in Examples 8, 11, and 14 with rabbits, andin Example 17 with horses; although the production of antibodies infowl, such as hens, is also known. For that purpose an immunogeniccomposition that includes at least one of the recombinant proteinsobject of this invention is used. Typically, an immunogenic compositionof this type may additionally include a pH buffer system and anadjuvant, such as the complete or incomplete Freund's adjuvant.According to this invention, a method for producing neutralizingantibodies for the venom of the spiders of the Loxosceles genus involvesinoculating a vertebrate with an effective amount sufficient to generateantibodies of an immunogenic composition similar to the one describedabove, that includes at least one of the recombinant proteins of thisinvention. The vertebrate, preferably a mammal, may be, among others, arabbit, a sheep, a goat or if possible, a horse, as illustrated inExample 17. Preferably, that method includes recovering the antibodiesfrom the animal, typically from blood serum or plasma. Preferably, thoseantibodies are neutralizers of the in vivo toxic effect of the wholevenom of the Loxosceles spider, preferably from species selected fromthe group that comprises L. boneti, L. reclusa and L. laeta.

An additional scope of this invention refers to the compositions thatcomprise the antibodies, or their antigen-binding fragments, thatneutralize the in vivo effect of the venom of the Loxosceles spider,produced by the method described above. Those compositions compriseantibodies generated against at least one of the recombinant proteinsobject of this invention. Preferably, the spider belongs to a speciesselected from the group consisting of L. boneti, L. reclusa or L. laeta.

Since the antibodies generated against the recombinant proteins objectof this invention, whether in an isolated form or as a mixture ofimmunogens, have proven to be neutralizers of the venom of theLoxosceles spider, they may be used as part of a pharmaceuticalcomposition to treat patients who have been bitten by Loxoscelesspiders, especially L. boneti, L. reclusa and L. laeta. Optionally, thepharmaceutical composition may include pharmaceutically acceptablecarriers like the ones described above.

According to this, another scope of this invention refers topharmaceutical compositions that comprise the neutralizing antibodies(or their fragments) produced by means of the methods described above,using one or more of the recombinant proteins object of this inventionas immunogens, where said composition neutralizes the toxic effect ofthe venom of the Loxosceles spider and is useful for the treatment ofindividuals who have been bitten by that Loxosceles spider.

In another scope, this invention refers to a method for treatingindividuals who need such treatment, particularly individuals that havebeen bitten by Loxosceles spiders, particularly L. boneti, L. reclusaand L. laeta, where that method consists of the administration of apharmaceutically effective amount of the pharmaceutical compositiondescribed above. The pharmaceutical composition may be locally orsystemically administered by intravenous, subcutaneous, intramuscular,vaginal, intraperitoneal, nasal, or oral routes to protect theindividuals from the toxic effect of Loxosceles spider venom.

The recombinant proteins object of this invention (SMDrLb, SMDrLr1,SMDrLr2, SMDrLl1, SMDrLl2, SMDrLb(H11K), and SMDrLb(E31K)), may also beused to generate an antigenic matrix when bound, either covalently or byhydrophobic or hydrophilic interactions, to a substrate such aspolyacrylamide, polyvinyl, activated aldehyde-agarose (U.S. Pat. Nos.5,904,922 and 5,443,976), sepharose, carboxymethyl cellulose or someother, in such a way that matrix is capable of binding specifically toeither antibodies (generated against the whole venom of Loxoscelesspiders or against those same venoms enriched with some of therecombinant proteins object of this invention, or against a mixture ofthose recombinant proteins object of this invention) or against theF(ab) or F(ab′)2 fragments obtained from the hydrolysis of suchantibodies, being useful for purifying those antibodies or F(ab) orF(ab′)2 fragments by immunoaffinity, so the use of the recombinantproteins object of this invention in the antigenic matrix and saidantigenic matrix are included in the scope of this invention. Thisimmunopurification of the antibodies or their fragments during theantivenom manufacturing process will help to decrease the proportion ofthe irrelevant exogenous protein administered to a patient bitten by aLoxosceles spider. Therefore, another aspect of this invention is acomposition that includes at least one of the recombinant proteinsobject of this invention, bound to a substrate, characterized by thefact that composition is capable of binding specifically to theantibodies generated against the venom of the Loxosceles genus spidersor against that venom enriched with at least one of the recombinantproteins object of this invention.

Another scope of this invention refers to the use of the recombinantproteins object of this invention (SMDrLb, SMDrLr1, SMDrLr2, SMDrLl1,SMDrLl2, SMDrLb(H11K) and SMDrLb(E31K)) indiagnosis. Through the use ofany of the recombinant proteins of this invention, it is possible togenerate specific monoclonal antibodies against an epitope present onlyin that recombinant protein or in the corresponding native toxin of thevenom of the homologous species of Loxosceles spiders, but absent in thetoxin of the venom of the other Loxosceles species. These recombinantproteins may be used as part of an antigenic matrix in which they arebound covalently or by hydrophobic or hydrophilic interactions to asubstrate, and that matrix may be used as part of a diagnostic device.By testing a sample of an individual supposedly bitten by a Loxoscelesspider, said device will be able to be used to detect the presence, in asample of antibodies generated, by the body of the individual,specifically against the homologous native toxin of the venom of aspecific Loxosceles spider, determining if the spider that bit theindividual belonged to that species of Loxosceles. This can be done witheach of the recombinant proteins object of this invention, which wouldhelp to determine which of the homologous species (L. boneti, L. reclusaor L. laeta, Peruvian variety) the spider that caused the bite belongsto, providing the attending physician with a tool for directing thetreatment specifically. A method to diagnose whether the animal that bitthe individual belongs to a particular species of Loxosceles spider willinclude putting the above-mentioned device in contact with a sample ofthe individual bitten. If they are present, the antibodies generatedspecifically against the natural toxin of the venom of the spider thatbit the individual will recognize and bind to one of the recombinantproteins of the device. An optional detection system may be used toreveal the presence of the antibodies of the sample bound to therecombinant protein of the device. This detection system may be based onimmune-enzymatic, immune-fluorescence or immune-chromatographic methods.

Materials and Methods

1. Spiders

The specimens of L. boneti were gathered by people trained to recognizethe Loxosceles genus in the communities of La Capilla and Corral deToros, municipality of Iguala, which are located in the central regionof the State of Guerrero, Mexico, according to the distributiondetermined by Hoffman (1976) and Gerstch (1983). To confirm this, 10female and 10 male spiders were sent to the Museum of Natural History ofNew York to Dr. Norman Platnich, who identified them as such. Thespecimens of L. reclusa were collected in Stillwater, Okla. by theinventors of this invention, while the specimens of L. laeta (Peruvianvariety) were collected in Lima, Peru.

2. Obtaining the Venom

The glands of the spiders were mechanically extracted, by pulling thechelicerae to uproot them. They were placed in an ammonium acetatebuffer 20 mM pH 4.7 and were macerated with a Teflon homogenizer (50venom apparatuses per mL); they were centrifuged for two minutes at14,000 rpm to remove the undesired solid residues and cellular remains;they were stored at −70° C. until their use.

Limited amounts of pure venom (different from the gland extractdescribed) were also obtained. For that purpose, we took advantage ofthe fact that when manipulated, some spiders secrete small amounts ofvenom; those were collected in microcapillary tubes.

3. Biochemical Tests

(a) Molecular Exclusion Gel Chromatography

A 170 cm long and 1.4 cm in diameter column was used. The resin selectedto pack the column was Sephadex G-75 (SIGMA CHEMICAL CO.) because itsexclusion limit is 70 kDa. The run buffer was ammonium acetate 20 mM, pH4.7. The flow speed in the experiment was of 48 mL h⁻¹ cm⁻².

72.56 mg of venom (3.5 mL) were applied, measured by absorbance at 280nm. Samples were collected every six minutes with an approximate volumeof 4.5 mL; the spectrophotometer (Beckman DU650i) was read at twowavelengths: 260 nm and 280 nm.

(b) Polyacrylamide Gel Electrophoresis (SDS-PAGE).

The proteins were separated from the venoms in polyacrylamide gels at12.5% in reducing conditions at constant current. The equipment used forthis method was the Mini Protean III (BIO-RAD). Different concentrationswere used for each sample subjected to electrophoresis.

For the reducing conditions, 2-mercaptoethanol was used at a finalconcentration of 2.5%. The prestained molecular weight markers (BioLabs,Inc.) were used as molecular weight standards. All the samples,including the molecular weight marker, were previously denaturalized ina water bath for 5 minutes. These were run at a constant current of 15mA, until the dye penetrated the separating gel; subsequently, thecurrent was increased to 25 mA. Once the gel run was finished, weproceeded to perform staining with Coomassie bright blue for one hour,and it was decolorated with a solution of 10% of acetic acid and 25% ofmethanol overnight with constant agitation.

(c) Ion-Exchange Chromatography (FPLC)

The column used was of the Mono S HR 5/5 type (Pharmacia LKB Biotech),which is a strong cation exchanger based on hydrophilic resins. The flowused for the runs was 1 mL/min. The buffers used were the following:

Initial buffer: Buffer A—Ammonium acetate 20 mM, pH 4.7.

Buffer limit: Buffer B—Ammonium acetate 20 mM, pH 4.7+2 M sodiumchloride.

Both buffers (filtrated through a 0.22 micron membrane) were run tocalibrate the column according to the distributor's specifications:

Once the column was balanced, the sample was injected. The sample waspreviously centrifuged for two minutes at 14,000 rpm, to clarify it(remove residues and/or precipitates).

The sensitivity of the detector was 0.2 AU, the flow rate was 1 mL/minand the gradient was from 0 to 2 M sodium chloride.

4. Activity of Sphingomyelinase D.

The measurement of this enzymatic activity was done with the Amplex RedSphingomyelinase Assay Kit (Molecular Probes) following the protocolsuggested by the manufacturer, using serial dilutions (1:1) startingfrom 1 μg/ml.

5. Measurement of Titers by ELISA of the Sera and in Vitro Determinationof Cross-Reactions

The titration of antibodies from sera was performed by Enzyme-LinkedImmuno Assays (ELISA). This assay was also used to observe possiblecross-reactions.

The ELISA assay consisted of:

1. Sensitization of 96-well plates for ELISA (Maxi sorp, NUNC™ Brandproducts) with a an antigen solution at a concentration of 5 μg/mL,reconstituted in 100 mM carbonate/bicarbonate pH 9.5.

100 μL were dispensed in each well up to column 11, since track 12served as negative control. The plate was incubated overnight at 4° C.

2. Once the incubation was completed, it was washed three times with 200μL of washing solution. This process had to be repeated each time weadvanced to the next step along the complete technique.

3. Afterwards, the unspecific protein binding sites were blocked with200 μL of blocking solution, for 2 hours at 37° C.

4. Step 2 is repeated.

5. Serial dilutions of the sera with an initial dilution of 1:30 in anELISA reaction buffer (indicated in the corresponding addendum) weremade. 100 μL of the ELISA reaction solution were added to each well, and50 μL/well of the anti-loxosceles serum dilution were mixed in column 1,to proceed to the 3× serial dilutions up to column 10 leaving columns 11and 12 as controls. It was incubated for one hour at room temperature.

6. Repeat step 2.

7. Then the second anti-rabbit antibody conjugated with the peroxidaseenzyme diluted to 1:1000 in an ELISA reaction solution was incubated,placing 100 μL/well. The incubation time was 1 hour at room temperature.

8. The reaction was revealed with 100 μL/well of ABTS substrate(Boehringer), incubating for 5 minutes at room temperature. After fiveminutes, the reaction was stopped with 25 μL of fluorhydric acid(Aldrich), and the plate was read in an ELISA plate reader (modelBIO-RAD 550) at 405 nm.

To determine the titers of the readings obtained, the sigmoid curveswere generated with the GraphPad Prism software (Version 2; GraphPadSoftware, Inc, San Diego, Calif.). The inflection point was calculatedby adjusting the experimental data for each venom and each antivenom bynonlinear regression of the sigmoid curves.

6. Western Blot Tests

This is a technique used to identify which mixture of proteins or theirfragments react to a determined antibody or antiserum. Western Blottests were performed according to the Mathews and Holde protocol (1998).

Polyacrylamide gels 12.5% were prepared and run in the usual way toseparate the proteic components of the venoms of L. boneti and L.reclusa. The amount of venom used for each assay was 30 μg per track.Once the gel run was completed, the transference to a nitrocellulosemembrane (solid substrate) was performed during one hour at constantcurrent (400 mA).

6. Western Blot Tests

This is a technique used to identify which proteins, or their fragments,within a mixture react to a particular antibody or antiserum. WesternBlot tests were performed according to the Mathews and Holde protocol(1998).

12.5% polyacrylamide gels were prepared and run in the usual way toseparate the proteic components of the venoms of L. boneti and L.reclusa. The amount of venom used for each assay was 30 μg per track.Once the gel run was completed, the transference to a nitrocellulosemembrane (solid substrate) was performed for one hour at constantcurrent (400 mA). For this purpose, a transference chamber undersemi-humid conditions was used (OWL) Once the transference to themembrane was completed, it was blocked overnight with constant stirringat room temperature in a 5% solution of skimmed milk/TBST, to preventthe unspecific binding of the antibodies. After this time, the membraneswere washed three times with TBST 1× (ten minutes each washing).Afterwards, It was incubated with the first antibody in 0.1% powderedskimmed milk (Carnation or Svelty/TBST) with constant stirring at roomtemperature for one hour (it was diluted according to the titre of theantibody). The dilutions used in this assay were 1:1000, 1:2500, and1:5000.

Once the incubation was completed, we performed three ten-minute washeseach one with TBST 1×. The second antibody was incubated for one hour atroom temperature with constant stirring in 0.1% skimmed milk/TBST. Ananti-rabbit coupled to alkaline phosphatase (ZYMED) was used as thesecond antibody.

After the incubation, it was washed three times with TBST for tenminutes; the TBST from the last wash was eliminated, and NTB-BCIPreaction buffer, which was left to react for five minutes, was added;the reaction was stopped with 5 mM EDTA.

7. Determination of the LD50

To determine the LD50 of SMDrLb and SMDrLr of the native venoms of L.reclusa and L. boneti, groups of 5 Balb/c mice weighing 18-20 g wereused. Variable amounts of the toxin were applied intraperitoneally,ranging from 0.6 to 17.57 μg of toxin per animal, and an LD50 of 2.55 μgof SMDrLb and of 6 μg of SMDrLr were found per mouse. The SMDrLb usedhad a concentration of 331 μg/mL (BCA method) and a sphingomyelinase Dactivity of 104.77 U/mL; the SMDLr had a concentration of 53.6 μg/mL anda sphingomyelinase D activity of 103.38 U/mL. The calculations were madeusing the GraphPad Prism software (Version 2; GraphPad Software, Inc,San Diego, Calif.). The venoms used had a concentration of 3,300 μg/mLof protein (BCA) and a sphingomyelinase D activity of 7.5 U/mg for L.boneti, and 5,800 μg/mL (BCA) and 9.05 U/mg for the venom of L. reclusa.

8. Dermonecrosis in Rabbits

The dermonecrotic activity was assessed in rabbits and determined asdescribed by Furlanetto et al. (1962a, b).

Different concentrations of the venom were used; they were diluted in0.2 mL of PBS buffer, pH 7.4, or in 0.2 mL of ammonium acetate 20 mM, pH4.7. These were intradermically injected into the backs of two rabbits.

In order to better illustrate this invention and its use, the followingspecific examples are provided to help the reader better understand thedifferent aspects of the practice of this invention. Since thesespecific examples are merely illustrative, in no case should thefollowing descriptions be considered to limit the scope of thisinvention:

EXAMPLE 1 Biochemical Characterization of the Venom of Loxosceles Boneti

To characterize the venom of the Loxosceles boneti spider, which isfound in Mexico, particularly in the states of Guerrero, Puebla andMorelos, an extract of the glands of the spider was obtained. Theextract was lyophilized and reconstituted in ammonium acetate 20 mM pH4.7; the formation of precipitate was observed. An SDS-PAGE analysis(reducing conditions) showed that the protein of interest was primarilypreserved in the supernatant, with which we continued to work. It wasseparated by molecular exclusion gel chromatography, and 4 main peakswere observed. (FIG. 1). Correspondingly, the samples were separatedinto 4 fractions, with fraction II containing the protein ofapproximately 32 KDa, which would presumably be sphingomyelinase (FIG.2). This fraction II was characterized by ion-exchange chromatography byFPLC to search for possible isoforms, as in the reported cases of L.reclusa and L. intermedia. Three main peaks corresponding to isoforms I,II and III (FIG. 3) were obtained. Of these, only isoforms I and IIshowed proven dermonecrotic activity on the skin of rabbits, asdescribed by Furlanetto et al. (1962a, b). The sphingomyelinase activityof fraction II (which comprises the 3 isoforms) was 25 U/mg. Each of thethree separated isoforms was dried by centrifugation in SAVANT and thensequenced in an automated Beckman LF3000 apparatus, using Edman'schemistry (Walsh et al., 1981). From isoform I, the first 35 amino acids(SEQ. ID. NO:1) were sequenced; from isoform II, the first 33 (SEQ. ID.NO:2), and from isoform III, the first 22 (SEQ. ID. NO:3). The enzymaticactivity for isoform I was 30.5 U/mg; for isoform II, it was 9.5 U/mg,and for isoform III, 0 U/mg.

EXAMPLE 2 Generation of Polyclonal Antibodies in Mammals Against L.Boneti Venom and Cross-Reaction with L. Recluse Venom

To generate antivenom antibodies for L. boneti, 2 rabbits wereinoculated in a 13-inoculation plan. In the first 9 inoculations, thegland extract of L. boneti was administered in increasing amounts from20 to 250 μg, while in the remaining inoculations, 60, 80 and 100 μg offraction II (mixture of the 3 isoforms), obtained from the molecularexclusion gel chromatography, were administered. Inoculations wereperformed every 10 days. A volume of 1 mL (in PBS) with incompleteFreund's adjuvant was injected intradermically in all of the casesexcept for the first, in which complete Freund's adjuvant was injected.

The sera obtained were titrated, obtaining titers of up to 30,000. Incross-reactions with the venom of L. reclusa, the titers were up to23,000. FIG. 4 shows a Western Blot of the venom of L. reclusa and L.boneti (separated by SDS-PAGE) revealed with the anti-L. boneti serum,where the affinity towards the 32.5 KDa component of both venoms isclearly shown.

EXAMPLE 3 Isolation of Partial Clones of the Necrotoxins of L. Boneti

To isolate the clones of the necrotoxins, the RNAm of the venom glandsof spiders of each species was isolated using the TRIZOL method (Gibco),following the protocol of the manufacturer. The kit 3′ RACE (Gibco) wasused to synthetize the first chain. 2 μL of the total RNAm(approximately 500 mg) extracted from glands, 4 μL of water, and 1 μL(10 pmoles) of the Adapter Primer (AP) oligonucleotide, which comprisesa tail of poly Ts (SEQ.ID. NO:4), were incubated for denaturalization at7° C. for 10 min and cooled immediately in ice. 2 μL of PCR 10× buffer,1 μL of dNTPs (10 mM), 2 μL of MgCl2 (25 mM) and 2 μL of DTT (0.1 M)were added, preincubating for 2-4 min at 42° C. 1 μL of ReverseTranscriptase Superscript (Invitrogen) was added immediately; it wasmixed and incubated for 50 min at 42° C. The enzyme was inactivated byincubation for 15 min at 70° C., and the mixtures were immediatelycooled in ice for 1 minute. 1 μL of RNAsa H was added and incubated for20 min at 37° C. and stored at −80° C.

For the polymerase chain reaction (PCR), a sample of the first chainreaction (2 μL) was taken, and to it were added a buffer of PCR 10× Mg²⁺(100 mM TRIS-HCl pH8.3, 500 mM KCl 15 mM MgCl₂), 200 μM dNTPs, 20 pmolesof the direct oligonucleotide (at 5′-3′ sense), 20 pmoles of the reverseoligonucleotide (at 3′-5′ sense) AUAP (GIBCO) (SEQ.ID. NO:5) and twounits of Taq DNA Polymerase (ROCHE) in a final volume of 50 μL. Thereaction was performed using a Perking Elmer 9600 thermocyclator withthe following protocol: Incubation of the mixture for 3 min. at 94° C.,followed by 25 cycles of three incubation steps: 1 min at 94° C., 90 secat 48° C. and 2 min at 72° C. The Taq DNA polymerase binds to the 3′extremes, a useful A to hybridize with the T of the 5′ extremes of thelinearized cloning vector pCR 2.1-TOPO (3.9 Kb).

For L. boneti, 2 oligonucleotides were designed based on the aminoacidic sequence determined common to isoforms I and II, the oligo Lb1(SEQ. ID. NO:6) corresponding to the sequence of amino acids 5 to 10 ofthe amino terminus of isoforms I and II, and the Lb-nested oligo(SEQ.ID.NO:7), which comprises the last base of aa 7, and those from 8to 12, and the first two bases of aa 13. The first was used for the PCRreaction while the second was used for a confirmatory PCR. In bothcases, the commercial AUAP oligo (GIBCO) (SEQ ID NO:5), which recognizesspecifically the sequence of the AP oligo of the same company, was used

The products of the PCR were purified in an gel extraction kit (ROCHE),following the directions of the manufacturer. They were afterwardshybridized with (and bound to) to the pCR 2.1-TOPO (3.9 Kb) (Invitrogen)cloning vector linearized by the TOPO isomerase, which shows a salient Tin both chains, obtaining the plasmid with the insert (product of PCR30). These constructions were used to transform the cells of E. coli XL1blue strain. The selection of the clones that comprised an insert wasperformed by plating the transforming cells in Petri dishes with LB/agarwith ampicillin in the presence of X-Gal, selecting the white coloniesto amplify the plasmids. Thirteen positive colonies were obtained.

Of the 13 positive colonies, only 7 contained an insert of the correctsize. The plasmidic DNAs were sequenced in both chains using fluorescentnucleotides in a Perkin Elmer Applied Biosystems apparatus (Foster City,Calif., USA), as described by the manufacturer. Of the 7 positiveclones, one was selected for having the most clearly and unequivocallyidentified sequence, clone 30-8, with an approximate size of 900 pb.

EXAMPLE 4 Expression of the Partial Clone of L. Boneti

In order to be able to express the recombinant protein obtained in theprevious example, the cDNA was subcloned in the pTrcHIS-TOPO(INVITROGEN) expression vector. With the purpose of placing the gene inphase in the plasmid, 2 oligonucleotides were designed: the direct one,incomplete Lb5′ (SEQ.ID.NO:22), from the codifying sequences of aminoacids 5 to 9 of the amino terminus of isoforms I and II (the first 5 ofthe insert cloned in the previous step), plus the recognizing site forthe enzyme BamHI and 3 more bases so that the enzyme binds; and thereverse oligo, Lb3′Sal I (SEQ.ID.NO:9), from the codifying sequence ofthe aa 275 and 279 (the last 5 of the carboxyl terminus of L. boneti'ssphingomyelinase D), plus the terminus codon, plus the recognizing siteof the enzyme Sal O and 4 more bases to bind the enzyme. With theplasmid of clone 30-8 as a template and using the designed direct andreverse oligos as primers, a PCR reaction was performed obtaining a DNAfragment that comprises the codifying sequence of the recombinantprotein with the 5 to 279 amino acids of the sphingomyelinase of L.boneti, with a BamH I cut site in the 5′ terminus, and a Sal I cut sitein the 3′ terminus.

Both the pTrcHIS-TOPO expression vector and the above mentioned PCRproduct were digested with the BamH I and Sal I enzymes and bound toobtain the pTrcHIS-TOPO 30-8 plasmid, being transformed inelectro-competent cells of E. coli XL1 blue. Three (3) positive coloniesthat carried the pTrcHIS-TOPO 30-8 plasmid; they had an insert of thecorrect size (as observed in a gel, after releasing the insert bydigesting the plasmid with BamH I and Sal I) and they were obtained andconfirmed by sequencing.

To express the recombinant protein, each of the 3 colonies obtained wascultured in 3 mL of LB medium in the presence of ampicillin 100 μg/mL at37° C. overnight with stirring, afterwards they were transferred to a500-mL LB flask with ampicillin. Once the culture reached an opticaldensity of 0.6 OD₆₀₀, expression was induced with 1 mM IPTG for 3 hoursat 37° C. with stirring. The cells were harvested by centrifugation(8,000 rpm, 15 min) and resuspended in 5 mL of A buffer (NaH2PO4 100 mM,TRIS-HCl 10 mM pH8 and Guanidinium Chloride 6M) and sonicated afterwardsfor 6 cycles of 30 sec each with intervals of 1 min in ice. Then, it wascentrifuged for 25 min at 10,000 rpm. The supernatat was run through anickel (Ni-NTA agarose) (Qiagen) column to purify the protein. Once thepurification was concluded, it was dialized against PBS to eliminate theguanidinium chloride, but the protein precipitated. The precipitatedprotein was quantified by BCA (PIERCE), but when tested as an immunogenin rabbits, it didn't yield good results. Based on this, we decided toplicate the protein in vitro.

To plicate the protein in vitro, it was solubilized by placing it in thepresence of 5M of guanidinium chloride plus 30 M of DTT for 2 hours atroom temperature. After this time, the protein was dialyzed against asolution with 2M of guanidinium chloride, 4 mM reduced glutathione(GSH), and 2 mM of oxidized glutathione (GSSG) in PBS 1× pH 47.4, for 1hour. These dialysis were repeated with decreasing concentrations ofguanidinium chloride, keeping those of GSH and GSSG constant, except inthe last dialysis step in which they were eliminated from the solution;the protein remained in PBS 1× pH 7.4, recovering 80% of the protein ina soluble form. The solubilized protein lacked sphingomyelinase anddermonecrotic activity, and the antibodies generated in rabbits withthis recombinant protein were able to recognize the recombinant proteinitself and the dermonecrotic component of the whole venom detected byWestern Blot; however, they were unable to neutralize the toxocity ofsaid venom.

EXAMPLE 5 Isolation of the Complete Clone with Complete L. Boneti.Sphingomyelinase D

In order to obtain a codifying sequence of the complete sphingomyelinaseD of L. boneti (i. e., from the first amino acid of the amino terminusregion up to the last in the carboxyl terminus region), we designedoligonucleotide Lb5′BAM HI (SEQ.ID.NO:8), which also includes thenecessary sequence for the Bam H1 site, the codifying codons of thefirst 4 aa of the amino terminus of L. boneti's sphingomyelinase D, plusthe next 5 amino acids already present in the original product of thePCR obtained from the 30-8 clone. A PCR reaction was carried out usingthe pTrcHIS-TOPO 30-8 plasmid as a template and the LB5′BamH1 oligo asdirect primer and the same Lb3′Sal I oligo (SEQ.ID.NO:9) as reverseprimer, obtaining a DNA fragment that comprises the complete codifyingsequence of L. boneti's sphingomyelinase, flanked by the cut sites ofBamH I and Sal I. This fragment was cloned in the TOPO 2.1 (3.9 Kb)plasmid, transforming competent cells of E. coli XL1 blue; 4 positivecolonies were obtained, of which the nucleotidic sequence of the insertwas proven by sequencing. A larger amount of plasmid was produced, andthe insert, which comprises the complete codifying gene (SEQ. ID. NO:10) of the active recombinant protein SMDrLb (SEQ. ID. NO: 11), wasreleased by digestion with the Bam HI and Sal I enzymes. The insert waspurified using a purification kit (ROCHE) and bound to the pQE30 vector,previously digested with the same restriction enzymes, obtaining vectorPQE30Lb-8c3.1.

EXAMPLE 6 Expression of the Complete SMDrLb Clone

The product of binding with the plasmid PQE30Lb-8c3.1 was used totransform competent cells of E. coli BL21, which, when plated, produced3 positive clones, confirming the presence of the DNA fragment thatcomprises the complete codifying sequence of L. boneti'ssphingomyelinase D, as could be corroborated by restriction analysiswith Bam HI and Sal I enzymes.

The clone used for the expression was called PQE30Lb-8c3.1. For thatpurpose, it was cultured at 37° C. in LB plus ampicillin up to an OD of0.6; it was later induced with 1 mM of IPTG for 3 hours. Again, theprotein was expressed in considerable amounts, but in inclusion bodies,whose sphingomyelinase D activity was negligible.

EXAMPLE 7 Controlled Expression of the Complete Clone of L. Boneti'sSphingomyelinase D

In order to control the expression level of the recombinant SMDrLbprotein, the clone PQE30Lb-8c3.1 was incubated in 50 mL of LB withampicillin at 37° C. overnight; the cells were transferred to a flaskwith 1L of the same medium. Once an OD₆₀₀ of 0.6 was reached, theculture was induced with IPTG (0.1 mM), and it was incubated for 16hours at a lower temperature (20-22° C.) with stirring. The cells wererecovered by centrifugation (10 min at 8,000 rpm). The cell package wasresuspended in 20 mL of PBS, sonicated for 6 cycles of 30 seconds atintervals of 1 min in ice, and centrifuged again for 25 min at 10,000rpm. A protein with the expected molecular weight (32 Kda) was foundboth in the supernatant and in the pellet

The soluble recombinant protein was purified by running the supernatantthrough an NiTA (nickel tri-nitrile-acetic, QIAGEN) column, where therecombinant protein attaches to the metal due to high affinity of the 6histidine tail. It was subsequently washed with 15 volumes of PBS andwith 10 volumes of PBS plus imidazole 25 mM, and finally eluted therecombinant protein with PBS plus 250 mM of imidazole. The elusion wascollected and dialyzed against PBS to eliminate the imidazole that wasstill present. The sphingomyelinase activity was measured and turned outto be 31.5 U/mg, i.e., practically the same as the activity found forthe native isoform I.

EXAMPLE 8 Production of Neutralizing Antibodies from the ActiveRecombinant Protein SMDrLb

With the active recombinant soluble protein SMDrLb produced as set forthin Example 7, two 3.5-Kg New Zealand rabbits were inoculated with ascheme of 8 inoculations at 10-day intervals, with increasing amounts ofthe recombinant protein, from 30 to 100 μg/rabbit of (active) solublerecombinant protein in PBS. Inoculations were applied intradermically ina final volume of 1 mL, with 0.5 mL of Freund's adjuvant, complete forthe first inoculation and incomplete for the subsequent inoculations.

Ten days after the eighth inoculation, the antibody titre was measured,and it was 22,800 for one rabbit and 29,300 for the other. The rabbitswere bled to death, and the serum of both rabbits was separated andmixed (50/50% in volume).

To determine the median effective dose (ED50) of the serum mixture, 3challenges were performed with different amounts of the same recombinantprotein SMDrLb, 7.8, 15.45 y 18.1 μg/mouse. For that purpose, groups of4 Balb-C mice weighing 18-20 g were used; they were injected with apre-incubated (30 min at 37° C.) mixture of the SMDrLb protein withincreasing amounts (from 100-200 μL) of the serum mixture of the 2inoculated rabbits in SS (NaCl 0.15 M), obtaining the following ED50;Mean while in a similar assay, 92 μg of the native venom ofpre-incubated L. boneti with increasing amounts of the homologous serum(anti-SMDLrLb) were applied, thus obtaining an ED50 of 149.6 μg permouse, which for the 92 μg are equivalent to 3LD50.

SMDrLb quantity ED50  7.8 μg 154.6 μl 15.5 μg 258.7 μl 18.1 μg 276.5 μl

The calculations were made using the GraphPad Prism software (Version 2;GraphPad Software, Inc, San Diego, Calif.).

EXAMPLE 9 Isolation of Complete Clones of L. Recluse Sphingomyelinase D

To isolate the clones of the necrotoxins, the total RNA of the venomglands of spiders was isolated using the TRIZOL reagent method (Gibco),following the protocol of the manufacturer. Kit 3′ RACE (Gibco) was usedto synthetize the first chain. 2 μL of the total RNA (approximately 500ng) extracted from glands, 4 μL of water, and 1 μL (10 pmoles) of theAdapter Primer (AP) oligonucleotide, which comprises a tail of poly-Ts(SEQ.ID. NO:4), were incubated for denaturalization at 70° C. for 10 minand cooled immediately in ice. 2 μL of PCR 10× buffer, 1 μL of dNTPs (10mM), 2 μL of MgCl2 (25 mM) and 2 μL of DTT (0.1 M) were added,preincubating them for 2-4 min at 42° C. 1 μL of Reverse Transcriptase(Invitrogen) was added immediately; it was mixed and incubated for 50min at 42° C. The enzyme was inactivated by incubation for 15 min at 70°C., and the compound was immediately cooled in ice for 1 minute. 1 μL ofRNAsa H was added, and it was incubated for 20 min at 37° C. and storedat −80° C.

In order to do the polymerase chain reaction (PCR), a sample was taken(2 μL) of the reaction made with the first chain and it was added PCRbuffer 10× Mg²⁺ (100 mM TRIS-HCL pH 8.3, 500 mM KCl 15 mM MgCl₂,), 200μM dNTPs, 20 μmoles of the direct oligonucleotide (in the 5′-3′ sense).In this case we used the same LB5′Bam H1 oligonucleotide (SEQ. ID. NO:8)which includes in addition to the necessary sequence for the BamH1 site,the codifying codons of the first 9 AAs of the amino terminus of L.reclusa sphingomyelinase D. 20 μmoles of the reverse oligonucleotide (inthe 3′-5′ sense), the reverse Lb3?Sal I (SEQ. ID. NO: 9) oligo and twounits of the Taq DNA Polymerase (New England Biolabs, Beverly, Mass.,USA) in a final volume of 50 μL. The reaction was performed using aPerkin Elmer 9600 thermocyclator with the following protocol: Incubationof the mixture for 3 min. at 94° C., followed by 25 cycles of threeincubation steps: 1 min at 94° C., 90 sec at 48° C., and 2 min at 72° C.At the end, a step of 10 min at 72° C. The Taq DNA polymerase binds tothe 3′ extremes, a useful A to hybridize with the T of the 5′ extremesof the cloning vector pCR 2.1 TOPO (3.9 Kb).

The products of the PCR were purified in an extraction kit (ROCHE),following the directions of the manufacturer. They were subsequentlyhybridized with (bound to) the pCR 2.1-TOPO (3.9 Kb) (Invitrogen)cloning vector linearized by the TOPO isomerase, which shows a salient Tin both chains, obtaining the plasmids pCR 2.1-TOPO Lr1 and pCR 2.1-TOPOLr2 that comprise the insert produced by PCR, Lr1 and Lr2, respectively.These constructions were used to transform the cells of E. coli XL1 bluestrain. The selection of the clones that comprised an insert wasperformed by plating the transforming cells in Petri dishes with LB/agarwith ampiciline in the presence of X-Gal, selecting the white coloniesto amplify the plasmids. Five positive colonies were obtained.

Of the 5 positive colonies, only 2 (Lr1 and Lr2) contained an insert ofthe correct size. The plasmidic DNAs were sequenced in both chains usingfluorescent nucleotides in a Perkin Elmer Applied Biosystems apparatus(Foster City, Calif., USA), as described by the manufacturer. The 2positive clones were selected because they had the sequence most clearlyidentified as sphingomyelinase D and with variations among them (90%identity). Both clones, with an approximate size of 900 pb, comprisedrespectively the complete codifying sequences of two isoforms of thesphingomyelinase D of L. reclusa (SEQ. ID. No: 12 and SEQ. ID. No: 14)whereas the codified proteins (SMDrLr1 and SMDrLr2) had the amino acidicsequences SEQ.ID. No: 13 y SEQ.ID. No: 15. For each of them, the insert,which comprises the gene of the corresponding SMDrLr, was released bydigestion with the enzymes Bam HI and Sal I. Each insert was purifiedusing a purification kit (ROCHE) and bound to the pQE30 vector,previously digested with the same restriction enzymes, obtaining vectorsPQE30Lr1 and PQE30Lr2.

EXAMPLE 10 Controlled Expression of the Complete Clones of SMDrLr

In order to control the expression level of the recombinant protein(both SMDLr1 and SMDrLr2), the cells of E. coli BL21 transformed withvector PQE30Lr1 or PQE30Lr2 were incubated in 3 mL of LB with ampicillinat 37° C. overnight; they were transferred to a flask with 100 mL of thesame medium containing 1 mM of IPTG, and they were incubated for 16hours at a temperature of 20-22° C. The cells were recovered bycentrifugation (10 min at 10,000 rpm). The cell package was resuspendedin 5 mL of PBS, sonicated for 6 cycles of 30 seconds at intervals of 1min in ice, and centrifuged again for 25 min at 10,000 rpm. A proteinwith the expected molecular weight (32 Kda) was found both in thesupernatant and in the pellet.

The soluble recombinant proteins SMDrLr1 and SMDrLr2 were purified byrunning the supernatant through a NiTA (nickel tri-nitrile-acetic,QIAGEN) column, where the recombinant protein attaches to metal due tothe high affinity of the 6 histidine tail. They were subsequently washedwith 10 volumes of PBS and with 10 volumes of PBS plus imidazole 25 mM,and finally eluting the recombinant protein with PBS plus 250 mM ofimidazole. The elusion was collected and dialyzed against PBS toeliminate the imidazole that was present. The sphingomyelinase activitywas measured and turned out to be 27.2 U/mg for SMDrLr1, i.e.,practically the same as the activity found for the native isoform and11.47 U/mg for SMDrLr2.

EXAMPLE 11 Production of Neutralizing Antibodies from the ActiveRecombinant Protein SMDrLr1

One of the recombinant proteins was selected: SMDrLr1. With the proteinproduced as set forth in Example 10, two 3.5-Kg New Zealand rabbits wereinoculated with a scheme of 8 inoculations at 10-day intervals withincreasing amounts of the recombinant protein, from 30 to 100 μg/rabbitof (active) soluble recombinant protein in PBS. Inoculations wereapplied intradermically in a final volume of 1 mL, with 0.5 mL ofFreund's adjuvant, complete for the first inoculation and incomplete forthe subsequent inoculations.

The antibody titre was measured; it was 26,000 for one rabbit and 33,000for the other one. The rabbits were bled to death, and the serum of bothrabbits was separated and mixed (50/50% in volume).

To determine the median effective dose (ED50) of the serum mixture,groups of 4 Balb-C mice weighing 18-20 g were used; they were injectedwith a pre-incubated (30 min at 37° C.) mixture of the SMDrLr1 toxinwith increasing amounts of the serum mixture of the 2 inoculatedrabbits, in SS (NaCl 0.15 M).

The median effective dose of the anti-SMDrLr1 antivenom against theactive recombinant protein proved to be 165 μL for mice, with 12 μg ofprotein. The calculations were made using the GraphPad Prism software(Version 2; GraphPad Software, Inc, San Diego, Calif.); Mean while, in asimilar assay, 12 μg of the native venom of L. reclusa pre-incubatedwith increasing quantities of the homologous serum (anti-SMDrLr1), wereapplied to each mouse, and was found an ED50 of 175 μL per mouse with 12μg of homologous venom

EXAMPLE 12 Isolation of the Complete Clones of the CompleteSphingomyelinase D of L. Laeta

We proceeded as in Example 9, but using venom glands from L. laeta asthe source of transcripts. The direct oligo LI5′BamH1 (SEQ.ID.NO:16) andthe reverse oligo LI3′Sal I (SEQ.ID.NO:17) were used for the PCR. Thesewere designed based on the sequences reported for the Brazilian variety(Fernandes Pedrosa, et al., 2002), whose products were purified andselected in the same way, obtaining 2 clones with a clearly identifiedsequence and with variations between them (88% of identity); theseproducts comprised, respectively, the complete codifying sequences oftwo isoforms of the sphingomyelinase D of L. laeta (SEQ. ID. No: 18 andSEQ. ID. No: 20), while the codified proteins (SMDrLl1 And SMDrLl2) hadthe amino acidic sequences SEQ.ID. No: 19 and SEQ.ID. No: 21. Finally,they were subclonated in the plasmid pQE30, obtaining vectors PQE30Ll1and PQE30Ll2, ready for expression.

EXAMPLE 13 Controlled Expression of the Complete SMDrLl1 Clone

Two clones, SMDrLl1 and SMDrLl2, were selected for expression. Toachieve a controlled expression, so as to prevent all of the recombinantprotein from being expressed as inclusion bodies, we proceeded as inExample 10, but transforming the cells with vectors PQE30Ll1 andPQE30Lr2. The recombinant proteins thus produced had an activity of58.43 U/mg for SMDrLl1 and of 252 U/mg for SMDrLl2.

EXAMPLE 14 Production of Neutralizing Antibodies from the ActiveRecombinant Protein SMDrLl1

We proceeded in a manner similar to Example 11, but using the SMDrLl1protein from Example 13 to inoculate the animals. The titers reachedvalues of up to 34,300, and an ED50 of 200 μL per mouse with 12 μg ofSMDrLl1 and of 225 μL per mouse with 12 μg of the homologous venom wasfound.

EXAMPLE 15 Cross-Protection Assays

Cross-protection assays were successfully carried out challenging micewith 12 μg of L. boneti venom or SMDrLb protein; we found that an ED50of 112 μL of rabbit anti-SMDrLr serum per mouse, and 200 μL of the sameper mouse were enough to neutralize 100% of the toxic effect of thevenom. In the same way, mice were challenged with 12 μg of native venomof L. reclusa or SMDrLr1 protein, and it was found that between 160 and200 μL of rabbit anti-SMDrLb serum were enough to neutralize its toxiceffect.

EXAMPLE 16 Subcloning of Clones Lb1, Lr1, Lr2, and Ll2 with Histidinesin the Carboxyl Position

To demonstrate that the inclusion of the histidine tail in the aminoterminus of the recombinant SMD proteins object of this invention (aminoversions) has no significant effect on the effectiveness of the protein,some of the clones: Lb1, Lr1, Lr2 and Ll2 were subclonated in the pQE60plasmid that adds the histidine tail to the carboxyl terminus instead ofthe amino terminus. For this purpose, the same oligos were used: Lb5′BamH1 (for Lb1, Lr1 and Lr2) and Ll5′Bam H1 (for Ll2) as direct oligos, andLb3′ Bg1 II (SEQ.ID.NO: 23) and Ll3′ Bg1 II (SEQ.ID.NO: 24),respectively, as reverse oligos. The constructions were expressed in acontrolled manner in strains of E. coli BL21 in the same way as inExamples 7, 10, and 13. The recombinant proteins thus produced (carboxylversion), presented essentially the same SMD activities as the aminoversions.

EXAMPLE 17 Production of Antibodies in Horses, Against a Mixture ofSeveral of the Recombinant Proteins of the 3 Loxosceles Species

To further illustrate the ability of the recombinant proteins of thisinvention to generate neutralizing antibodies in vertebrates,particularly in mammals, this time a horse was selected for thegeneration of antibodies. At the same time, to illustrate thepossibility of using an immunogenic composition that comprises more thanone of the recombinant proteins of this invention, 4 of them which wereconsidered sufficiently representative were selected to neutralize thevenoms of at least 3 species of the Loxosceles spider, some in theiramino version and others in their carboxyl version. In this way, theimmunogenic composition was composed of 2 parts of SMDrLr1 (aminoversion or SMDrLr1-NH2), 2 parts of SMDrLb (carboxyl version orSMDrLb-COOH), 1 part of SMDrLl1 (amino version or SMDrLl1-NH2) and 1part of SMDrLl2 (carboxyl version or SMDrLl2-COOH).

Five horses that had never been immunized or had never had contact withany antigen related to the Loxosceles spider were selected andinoculated with the immunogenic composition mentioned in the previousparagraph. The inoculation was carried out over a period of 9 months,starting with a dose of 2.5 μg of the recombinant toxin mixture andending with 250 μg. The inoculations were performed at intervals of twoweeks, and Freund's adjuvant and alumina were used, alternately, asadjuvants. The inoculation was performed subcutaneously. Blood sampleswere taken from the horses at one-month intervals in order to measurethe antibody titers by immunoenzymatic assay.

The horses were bled at the end of the nine-month period, and the plasmawas mixed and processed to produce F(ab′)2 fragments by digestion withthe pepsin enzyme and its subsequent purification. The median effectivedose of the F(ab′)2 fragments was determined in a similar way to thatdescribed in Example 8. Fourteen (14) groups of 5 Balb-C mice weighing18-20 g were used. Two groups were immunized (one control and onetreated with F(ab′)2 fragments) for each of the following recombinantproteins (recombinant toxins) or venoms: SMDrLr1-NH2, SMDrLb-COOH,SMDrLl1-NH2, SMDrLl2-COOH, venom of L. boneti, venom of L. reclusa, andvenom of L. laeta For this purpose, they were injected with apre-incubated mixture (30 min at 37° C.) of 5LD50 of the recombinantprotein or venom, either with increasing amounts (from 100-200 μL) ofthe F(ab′)2 fragments obtained, in SS (NaCl 0.15 M) for the treatmentgroups, or with SS only for the control groups. From the ED50sdetermined by using the above-mentioned GraphPad Prism software, theamounts of neutralized toxin or venom per 1 mg of F(ab′)2 fragments(neutralizing ability) were calculated, and they are shown below:

μg of toxin or venom Recombinant toxin neutralized per mg of or naturalvenom F(ab′)2 fragments SMDrLb-COOH 251 SMDrLr1-NH2 235 SMDrLl1-NH2 55SMDrLl2-COOH 105 L. boneti venom 629 L. reclusa venom 587 L. laeta venom394

EXAMPLE 18 Obtaining Toxoids of SMDrLb

Based on the recently reported (Murakami et al., 2005) identification ofthe catalytic center of one of the isoforms of the sphingomyelinase D ofL. laeta, we decided to perform mutagenesis on the codifying DNA ofSMDrLb (SEQ. ID.NO:10), a mutant in the codifying codon of the histidineresidue in position 11, and the other one in the codifying codon for theglutamic acid residue in position 31, both counted in the matureprotein. The first mutant obtained, designated SMDrLb(H11K), consistedof substituting the histidine in position 11 for a lysine; it has aSEQ.ID.NO:27 for its codifying sequence and SEQ.ID.NO:28 for the matureexpressed protein. The second mutant, designated SMDrLb(E31K), consistedof substituting the glutamic acid in position 31 for a lysine; it has aSEQ.ID.NO:29 for its codifying sequence and a SEQ.ID.NO:30 for theexpressed mature protein.

To obtain the mutants, clone PQE30Lb-8c3.1 and the QuickChange directedmutagenesis kit (QuickChange Site-Directed Mutagenesis Kit) (Stratagene,La Jolla, Calif., USA) were used, following the protocol recommended bythe company (Papworth, C. et al., 1996).

The two mutants, SMDrLb(H11K) and SMDrLb(E31K), incorporated in thepQE30 vector were isolated and expressed in a controlled way in the E.coli BL21 strain, in a way similar to the one described for SMDrLb inExample 7.

Both proteins were successfully expressed in a soluble form (and part ofthem as inclusion bodies). Their sphingomyelinase D enzymatic activitywas analyzed by the method described previously, and its result wasnull. Additionally, dermonecrosis tests were performed on rabbits, whichdemonstrated that they lack this effect.

To determine if these enzymatically inactive and non-dermonecroticversions expressed in a soluble form were capable of generatingneutralizing antibodies for the active SMDrLb or the venom of L. boneti,for each of the mutants, two 3.5-kg New-Zealand rabbits were inoculatedwith a scheme of 8 inoculations, at 10-day intervals, with increasingamounts of the recombinant protein, from 30 to 100 μg/rabbit ofrecombinant soluble protein (inactive) in PBS. Inoculations wereperformed intradermically in a final volume of 1 mL, with 0.5 mL ofFreund's adjuvant, complete for the first inoculation and incomplete forthe subsequent inoculations.

In both cases, titers that varied from between 25,200 and 31,500 wereobtained. For each case, the rabbits were bled to death, and the serumof both rabbits was separated and mixed (50/50% in volume).

To determine the median effective dose, ED50, of the serum mixture ofeach case, groups 4 of Balb-C mice weighing 18-20 g were used. They wereinjected with a pre-incubated (30 min at 37° C.) mixture of 12 μg ofSMDrLb toxin with increasing amounts of the serum mixture of the 2inoculated rabbits, in SS (NaCl 0.15 M).

From the ED50s determined using the GraphPad Prism software, the amountsof neutralized toxin or venom per 1 mL of each of the sera (neutralizingability) were calculated, and they are shown below:

μg of toxin or venom neutralized per mL of serum Recombinant toxin anti-anti- or natural venom SMDrLb(H11K) SMDrLb(E31K) SMDrLb-COOH 58.5 53.8L. boneti venom 162.5 151.1

REFERENCES

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1.-56. (canceled)
 57. A pharmaceutical composition for passiveimmunization comprising polyclonal antibodies or active fragmentsthereof, directed to one or more of the group of the following antigensof genus Loxosceles, and their functional variants with at least 90% ofidentity: SMDrLb1 of SEQ ID NO: 11, SMDrLr1 of SEQ ID NO: 13, SMDrLI1 ofSEQ ID NO: 19, SMDrLI2 of SEQ ID NO:
 21. 58. The pharmaceuticalcomposition of claim 57 where the polyclonal antibodies are F(ab′)₂antibody fragments.
 59. A method of treatment for envenomation thatconsist in administrating the pharmaceutical composition of claim 57,where such envenomation is caused for one or more of the spiders ofgenus Loxosceles selected form the group consisting of: L. laeta, L.boneti, and L. reclusa.
 60. The method of treatment for envenomationthat consist in administrating the pharmaceutical composition of claim59, where such envenomation is caused for one or more of the spiders ofgenus Loxosceles selected form the group consisting of: L. laeta, L.boneti, and L. reclusa.
 61. An immunogenic pharmaceutical compositioncomprising a mixture of one of more of the following recombinantantigens of genus Loxosceles: SMDrLb1 SEQ ID NO: 11, SMDrLr1 SEQ ID NO:13, SMDrLI1 SEQ ID NO: 19, SMDrLI2 SEQ ID NO:
 21. 62. A method toproduce polyclonal antibodies comprising the following steps: i) mixingthe following antigens in proportion 2:2:1:1 respectively: SMDrLb1 SEQID NO: 11, SMDrLr1 SEQ ID NO: 13, SMDrL11 SEQ ID NO: 19, SMDrL12 SEQ IDNO:
 21. ii) immunizating of a non-human animal with the pharmaceuticalcomposition of claim 61, iii) obtaining blood serum and the antibodyactive fraction, where said fraction is capable of neutralize the venomof one or more of the spiders L. laeta, L.boneti and L. reclusa.
 63. Themethod of claim 62 where immunization step (ii) is carried out in Eqquscaballus.